Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

WB

Use a concentration of 0.5 - 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 75 kDa).Can be blocked with Human MTA2 peptide (ab6243).

IHC-Fr

Use at an assay dependent concentration. PubMed: 18413351

IHC-P

Use at an assay dependent concentration.

ICC/IF

Use a concentration of 10 µg/ml.

ELISA

Use at an assay dependent concentration.

IP

Use a concentration of 5 µg/ml.

Target

RelevanceThe p53 tumor suppressor gene integrates numerous signals that control cell life and death. There are several proteins that are involved in the p53 pathway, including Chk2, p53R2, p53AIP1, Noxa, PIDD, and MTA2. The transcriptional activity of p53 is modulated by protein stability and acetylation. MTA2, also termed MTA1L1, was found to be a subunit of nucleosome remodeling and deacetylating (NuRD) complex. MTA2 modulates the enzymatic activity of the histone deacetylase complex and its expression reduces the levels of acetylated p53. Deacetylation of p53 by MTA2 represses p53 dependent transcriptional activation and modulates p53 mediated cell growth arrest and apoptosis. MTA2 is ubiquitously expressed in human tissues.

ab8106 (2µg/ml) staining MTA2 in human ileum using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining.Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Immunoprecipitation - Anti-MTA2 antibody (ab8106)

MTA2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to MTA2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab8106.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 77kDa: MTA2